Structure-Based Design of AG-946, a Pyruvate Kinase Activator.

Pyruvate kinase (PK) is the enzyme that catalyzes the conversion of phosphoenolpyruvate and adenosine diphosphate to pyruvate and adenosine triphosphate in glycolysis and plays a crucial role in regulating cell metabolism. We describe the structure-based design of AG-946, an activator of PK isoforms, including red blood cell-specific forms of PK (PKR). This was designed to have a pseudo-C2-symmetry matching its allosteric binding site on the PK enzyme, which increased its potency toward PKR while reducing activity against off-targets observed from the original scaffold.

Leveraging Structure-Based Drug Design to Identify Next-Generation MAT2A Inhibitors, Including Brain-Penetrant and Peripherally Efficacious Leads.

Inhibition of the -adenosyl methionine (SAM)-producing metabolic enzyme, methionine adenosyltransferase 2A (MAT2A), has received significant interest in the field of medicinal chemistry due to its implication as a synthetic lethal target in cancers with the deletion of the methylthioadenosine phosphorylase (MTAP) gene. Here, we report the identification of novel MAT2A inhibitors with distinct properties that may enhance their utility in treating patients. Following a high-throughput screening, we successfully applied the structure-based design lessons from our first-in-class MAT2A inhibitor, , to rapidly redesign and optimize our initial hit into two new lead compounds: a brain-penetrant compound, , and a potent, but limited brain-penetrant compound, .

Distinct Hepatic PKA and CDK Signaling Pathways Control Activity-Independent Pyruvate Kinase Phosphorylation and Hepatic Glucose Production.

Pyruvate kinase is an important enzyme in glycolysis and a key metabolic control point. We recently observed a pyruvate kinase liver isoform (PKL) phosphorylation site at S113 that correlates with insulin resistance in rats on a 3 day high-fat diet (HFD) and suggests additional control points for PKL activity. However, in contrast to the classical model of PKL regulation, neither authentically phosphorylated PKL at S12 nor S113 alone is sufficient to alter enzyme kinetics or structure.

Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers.

Hydrogen bonding networks tune proton-coupled redox steps during the enzymatic six-electron conversion of nitrite to ammonia.

Multielectron multiproton reactions play an important role in both biological systems and chemical reactions involved in energy storage and manipulation. A key strategy employed by nature in achieving such complex chemistry is the use of proton-coupled redox steps. Cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron seven-proton reduction of nitrite to ammonia.

Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity.

Anaerobic degradation of the environmental pollutant toluene is initiated by the glycyl radical enzyme benzylsuccinate synthase (BSS), which catalyzes the radical addition of toluene to fumarate, forming benzylsuccinate. We have determined crystal structures of the catalytic α-subunit of BSS with its accessory subunits β and γ, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSSα has the common glycyl radical enzyme fold, a 10-stranded β/α-barrel that surrounds the glycyl radical cofactor and active site.

Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence of interactions across the dimeric interface.

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, conducts the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by "boosts" and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to the properties of ecNrfA.

Laue crystal structure of Shewanella oneidensis cytochrome c nitrite reductase from a high-yield expression system.

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "small tetraheme c" replaces the wild-type signal peptide.

Ovariectomy in grasshoppers increases somatic storage, but proportional allocation of ingested nutrients to somatic tissues is unchanged.

Reduced reproduction increases storage and extends lifespan in several animal species. The disposable soma hypothesis suggests this life extension occurs by shifting allocation of ingested nutrients from reproduction to the soma. A great deal of circumstantial evidence supports this hypothesis, but no direct tracking of nutrients has been performed in animals that are long-lived because of direct reduction in reproduction.

Allocation of nutrients to somatic tissues in young ovariectomized grasshoppers.

The disposable soma hypothesis predicts that when reproduction is reduced, life span is increased because more nutrients are invested in the soma, increasing somatic repair. Rigorously testing the hypothesis requires tracking nutrients from ingestion to allocation to the soma or to reproduction. Fruit flies on life-extending dietary restriction increase allocation to the soma "relative" to reproduction, suggesting that allocation of nutrients can be associated with extension of life span.